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Molecular Cancer Research 3:63-70 (2005)
© 2005 American Association for Cancer Research

Cancer Genes and Genomics

Transactivation Functions of the Tumor-Specific HMGA2/LPP Fusion Protein Are Augmented by Wild-Type HMGA21

Koen R.M.O. Crombez, Els M.R. Vanoirbeek, Wim J.M. Van de Ven and Marleen M.R. Petit

Laboratory for Molecular Oncology, Department of Human Genetics, University of Leuven and Flanders Interuniversity Institute for Biotechnology, Leuven, Belgium

Requests for reprints: Wim J.M. Van de Ven, University of Leuven and Flanders Interuniversity Institute for Biotechnology, Department of Human Genetics, Herestraat 49 bus 602, B-3000 Leuven, Belgium. Phone: 32-16-34-60-80 or 32-16-34-59-87; Fax: 32-16-34 60 73. E-mail: wim.vandeven{at}med.kuleuven.ac.be


   Abstract
Top
 Notes
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
The gene encoding the architectural transcription factor HMGA2 is frequently rearranged in several benign tumors of mesenchymal origin. The lipoma preferred partner (LPP) gene is the most frequent translocation partner of HMGA2 in a subgroup of lipomas, which are benign tumors of adipose tissue. In these lipomas, HMGA2/LPP fusion transcripts are expressed, which encode for the three AT-hooks of HMGA2 followed by the two most carboxyl-terminal LIM domains (protein-protein interaction domains) of LPP. Identical fusion transcripts are also expressed in other benign mesenchymal tumors. Previous studies revealed that the LIM domains of LPP have transcriptional activation capacity in GAL4-based luciferase reporter assays. Here, we show that the HMGA2/LPP fusion protein retains the transactivation functions of the LPP LIM domains and thus functions as transcription factor. The HMGA2/LPP fusion protein activates transcription from the well-characterized PRDII element, which is a part of the IFN-ß enhancer and which is known to bind to HMGA2. We also show that HMGA2/LPP activates transcription from the BAT-1 element of the rhodopsin promoter, a HMGA1-binding element. HMGA1 is a closely related family member of HMGA2. Finally, in a number of lipomas, HMGA2/LPP and HMGA2 are coexpressed, and HMGA2 augments the transactivation functions of HMGA2/LPP. These results support the concept that the transactivation functions of the novel HMGA2/LPP transcription factor contribute to lipomagenesis.

Key Words: High mobility group A • lipoma preferred partner • mesenchymal tumor • LIM domain • luciferase reporter assay


   Introduction
Top
 Notes
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
High-mobility group A2 (HMGA2) is a member of the HMGA family of architectural transcription factors (1, 2). The HMGA protein family comprises four members, HMGA1a, HMGA1b, and HMGA1c, which are encoded by alternative splice products of the HMGA1 gene, and HMGA2, which is encoded by a different gene. The HMGA1 and HMGA2 proteins contain three DNA-binding domains, so-called AT-hooks, at their amino terminal, followed by an acidic carboxyl-terminal tail. AT-hooks modulate chromatin conformation by binding to AT-rich stretches in the narrow minor groove of DNA (3). HMGA proteins do not have intrinsic transcriptional activation capacity, but instead play a role in the formation and stabilization of enhanceosomes in enhancer or promoter regions, thus acting as architectural transcription factors (4, 5). This has been extensively studied in the context of the regulation of expression of the IFN-ß gene. It was shown that the accurate performance of the IFN-ß transcriptional switch depends on the ordered acetylation of the HMGA1 protein by PCAF/GCN5 and CREB-binding protein. Acetylation of HMGA1 by CREB-binding protein destabilizes the enhanceosome, whereas acetylation of HMGA1 by PCAF/GCN5 potentiates transcription by stabilizing the enhanceosome and preventing acetylation by CREB-binding protein (6). Concerning HMGA2, it was recently shown that HMGA2 modulates the activity of the DNA repair gene ERCC1 (7) and of the cyclin A gene (8) by binding to the promoters of these genes.

HMGA2 is highly expressed during embryonic and fetal stages of mouse development, but is barely detectable in adult tissues (9). Homozygous inactivation of the Hmga2 gene in mice results in the pygmy phenotype (displaying, among others, a small size and a drastic reduction of body fat content), indicating that the HMGA2 protein has a role in embryonic cell growth and differentiation (10). Wild-type HMGA2 has been reported to be overexpressed in various malignant tumors, such as breast cancer (11) and pancreatic tumors (12). In contrast to the overexpression of wild-type HMGA2 in malignant tumors, the HMGA2 gene is rearranged in a variety of benign solid tumors mainly of mesenchymal origin, such as lipomas (13-15), uterine leiomyomas (13), pleomorphic adenomas of the salivary glands (13), endometrial polyps (16), fibroadenomas of the breast (17), and pulmonary chondroid hamartomas (13, 18). Most of these HMGA2 rearrangements have chromosomal breakpoints within the large third intron of the gene, resulting in chimeric transcripts containing exons 1 to 3 of HMGA2 fused to limited ectopic sequences of other genes and encoding more or less a truncated form of HMGA2 comprising its three DNA-binding domains. These HMGA2 translocation partner genes are highly variable in nature. The lipoma preferred partner (LPP) gene on chromosome 3q27-28, however, was identified as a frequent translocation partner of the HMGA2 gene, occurring in ~10% of all solitary lipomas (15). These tumors express fusion transcripts coding for the three DNA-binding domains of HMGA2, followed by the two carboxyl-terminal LIM domains of LPP (15). Identical fusion transcripts are also found in a subgroup of pulmonary chondroid hamartomas (19), in a parosteal lipoma (20), and in a case of soft tissue chondroma (21). It should be noted that two alternative HMGA2/LPP hybrid transcripts have been detected encoding a long form or a short form of the HMGA2/LPP fusion protein (Fig. 1). The long form consists of the three DNA-binding domains of HMGA2 followed by part of the proline-rich region and all three LIM domains of LPP. The transcript encoding this long form has been detected in only one exceptional case of a lipoma (15). The transcript encoding the short form of HMGA2/LPP, consisting of the three DNA-binding domains of HMGA2 followed by the two most carboxyl-terminal LIM domains of LPP, is almost invariably found in the primary tumors. Therefore, only the short form of HMGA2/LPP was studied here.



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  		FIGURE 1. Schematic representation of wild-type HMGA2 and LPP proteins and related fusion proteins expressed in lipomas and other benign tumors of mesenchymal origin. The LPP protein consists of a proline-rich region, which is followed by three LIM domains (protein interaction domains). HMGA2 consists of three amino-terminal DNA-binding domains and an acidic carboxyl-terminal tail domain. The two variants of HMGA2/LPP fusion proteins that occur in benign mesenchymal tumors are schematically represented. The long form has been detected in only one exceptional case of a lipoma. DBD, DNA-binding domain; AD, acidic domain; LIM, LIM domain; PRR, proline-rich region.

 
LPP belongs to the zyxin family of proteins, which consists of five members: zyxin (22), thyroid receptor interacting protein 6 (TRIP6; ref. 23), LPP (15), ajuba (24), and LIM domain containing 1 (LIMD1; ref. 25). All of these proteins are characterized by the presence of three LIM domains at their carboxyl terminal, preceded by a proline-rich region containing several protein interaction domains. Zyxin family members have been proposed to be scaffolding proteins involved in signal transduction from sites of cell adhesion to the nucleus. The LPP protein localizes to focal adhesions and to cell-cell contacts (26). In addition, LPP can be transiently translocated to the nucleus (26). At cell adhesions, the LPP protein interacts with {alpha}-actinin and vasodilator-stimulated phosphoprotein (VASP), suggesting a role for LPP in certain aspects of cell motility and actin dynamics. Recently, LPP was found to be highly expressed in smooth muscle (27, 28), and a role for LPP in regulating cell motility was proposed (27). In the nucleus, LPP harbors a significant transcriptional activation capacity, suggesting that it is directly involved in the regulation of gene transcription (26).

Previous studies revealed that the LIM domains of LPP have transcriptional activation capacity in GAL4-based luciferase reporter assays. Here, we investigated whether the tumor-specific HMGA2/LPP fusion protein, which contains the two most carboxyl-terminal LIM domains of LPP, retains the transactivation functions of the LPP LIM domains. Therefore, reporter assays were done using constructs containing motifs to which HMGA2 and/or HMGA1 can specifically bind and from which the fusion protein might drive luciferase expression. We show that the HMGA2/LPP fusion protein is able to directly transactivate expression from these motifs. We also investigated coexpression of the rearranged HMGA2/LPP and the wild-type HMGA2 allele in different primary human lipomas as well as corresponding cell lines, and whether coexpression of HMGA2 affects the transcriptional activity of the tumor-specific HMGA2/LPP fusion protein.


   Results
Top
 Notes
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 Acknowledgements
 References
 
The Two Carboxyl-Terminal LIM Motifs of LPP Function as a Transactivation Domain
Previous studies revealed that LPP has transactivation functions and that the three LIM domains of LPP contribute to this transcriptional activation capacity (26). However, nothing is known about the tumor-specific HMGA2/LPP fusion protein, apart from its localization in the nucleus (26). The first aim of this study was to investigate whether the tumor-specific HMGA2/LPP fusion protein possesses transactivation capacity. Because the most frequent form of the HMGA2/LPP fusion protein contains only two LIM domains of LPP (short form), it was first determined whether these two LIM motifs function as a transactivation domain. In what follows, "HMGA2/LPP fusion protein" should be understood as the short form. To investigate this, the following GAL4-based transcriptional activation assay was done. An expression construct encoding the GAL4 DNA-binding domain (GAL4DBD) fused to the two most carboxyl-terminal LIM domains of LPP (GAL4DBD-LPP-LIM2/3) was transfected into HEK 293 cells together with a luciferase reporter plasmid, containing the luciferase gene under the control of a minimal promoter containing five consecutive binding sites for the GAL4 DNA-binding domain (26). Expression of GAL4DBD-LPP-LIM2/3 was assayed by Western blot analysis (data not shown). We measured the luciferase enzymatic activity in cell lysates, which is correlated to the level of expression of the reporter gene. We found that GAL4DBD-LPP-LIM2/3 enhanced the luciferase activity about 50-fold, as compared with the activity of the GAL4 DNA-binding domain alone (Fig. 2). Therefore, the two most carboxyl-terminal LIM motifs of LPP function as a transactivation domain.



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  		FIGURE 2. The two most carboxyl-terminal LIM domains of LPP have transcriptional activation capacity. A DNA construct expressing GAL4DBD-LPP-LIM2/3 was cotransfected in HEK 293 cells along with GAL4-regulated luciferase reporter DNA, and cell lysates were assayed for luciferase activity 48 hours after transfection. Luciferase activity is reported relative to the control, GAL4DBD. Average of three independent experiments that were carried out in duplicate.

 
The HMGA2/LPP Fusion Protein Functions as a Transcriptional Activator
Because the tumor-specific HMGA2/LPP fusion protein contains the AT-hooks of HMGA2, which are DNA-binding domains, followed by the two most carboxyl-terminal LIM domains of LPP, which, as shown above, have transcriptional activation capacity, the HMGA2/LPP fusion protein contains all of the necessary ingredients to act as a genuine transcription factor. To investigate whether the HMGA2/LPP fusion protein indeed can act as a transcription factor, we did several luciferase reporter assays. However, instead of using GAL4-based reporter constructs, we used reporter constructs that contain AT-hook binding sites to which the AT-hooks of HMGA2 and/or HMGA1 can bind, as was previously shown.

We first used the PRDII TKluc reporter (29), which contains two copies of the well-characterized PRDII element of the virus-inducible enhancer of the IFN-ß gene. The PRDII element binds nuclear factor {kappa}B in the major groove and the HMGA1 or HMGA2 protein in the minor groove of its central AT-rich region (29, 30). There, HMGA proteins do not have direct transcriptional activation capacity by themselves, but their binding to the PRDII element has been shown to potentiate the transcriptional activation by the nuclear factor {kappa}B protein (29, 30). We used the PRDII TKluc reporter to investigate the transcription factor properties, if any, of the HMGA2/LPP fusion protein because it was shown that HMGA2 binds to the PRDII elements with high affinity, suggesting that also the HMGA2/LPP fusion protein might bind to them. Upon cotransfection of a fixed amount of PRDII TKluc reporter DNA with increasing amounts of HMGA2/LPP expressing DNA (pHMGA2/LPP) in HEK 293 cells, we observed dose-dependent activation of luciferase gene expression (Fig. 3A). These results indicate that in contrast to wild-type and truncated HMGA2 (only AT-hooks; ref. 31), the HMGA2/LPP fusion protein possesses direct transactivation functions. Moreover, these results suggest that HMGA2/LPP, similar to wild-type and truncated HMGA2, is able to recognize and bind to the PRDII element. Indeed, when applying the same conditions but using a reporter construct with a mutant PRDII element (mPRDII TKluc), which is not bound by wild-type HMGA2 (29), no increase in luciferase activity was detected (Fig. 3B). Together with our findings regarding the transactivation function of the two most carboxyl-terminal LIM domains of LPP, the results suggest that the observed activation from the PRDII TKluc reporter by the HMGA2/LPP fusion protein is most likely due to the binding of the fusion protein to the PRDII element via its HMGA2 AT-hooks, and to the LIM-containing transactivation domain. Expression of the HMGA2/LPP fusion protein was verified by Western blot analysis and is shown together with wild-type HMGA2 (Fig. 3F).



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  		FIGURE 3. The HMGA2/LPP fusion protein possesses transcriptional activation capacity. A and B. HEK 293 cells were transfected with 200 ng of PRDII TKluc (A) or mPRDII TKluc (B) reporter plasmid DNA, together with increasing quantities of pHMGA2/LPP [50-300 ng per well, in steps of 50 (A) or 100 ng (B)]. The total DNA amount for transfection was kept the same in each well (500 ng) by normalizing with pcDNA3 vector DNA. Forty-eight hours after transfection, cells were harvested and assayed for luciferase activity. Activity is shown relative to basal PRDII TKluc or mPRDII TKluc luciferase activity (Mock). For each experiment, luciferase activity was determined in duplicate wells. Columns, means of at least three independent experiments; bars, SE. C and D. HEK 293 cells were transfected with 200 ng of pbRho-130 (C) or pbRho-130 mutHMGI (D) reporter plasmid DNA, together with increasing quantities of pHMGA2/LPP [50-300 ng per well, in steps of 50 ng (C) or 100 ng (D)]. The experiments were done as described in A and B. E. HEK 293 cells were transfected with 200 ng of pbRho-130 reporter DNA and 300 ng of LPP-LIM2/3 expressing vector DNA. The experiments were done as described in A and B. F. Western blot analysis of cell lysates of HEK 293T cells transfected with DNA of an HMGA2/LPP-, HMGA2-, or LPP-expressing construct.

 
Because the PRDII element was originally discovered as a HMGA1 recognition element (30, 32, 33), we evaluated whether the HMGA2/LPP fusion protein could also activate transcription from other known HMGA1-binding elements. To test this issue, we used a reporter construct with the proximal promoter of the rhodopsin gene to drive luciferase gene expression (pbRho-130; ref. 34). This proximal promoter contains a discrete HMGA1-binding site that overlaps with the BAT-1 cis element, which is the binding site for Crx, a paired-like homeodomain transcription factor essential for photoreceptor function. Upon cotransfection of a fixed amount of pbRho-130 reporter DNA with increasing amounts of pHMGA2/LPP in HEK 293 cells, we observed dose-dependent activation of the rhodopsin promoter-reporter (Fig. 3C). Therefore, HMGA2/LPP activates transcription from the pbRho-130 reporter. Upon application of the same conditions on the reporter construct containing a mutant HMGA1-binding element (pbRho-130 mutHMGI), activation of luciferase gene expression was significantly reduced compared with the reporter harboring the wild-type binding elements (Fig. 3D). These results suggest that the binding of HMGA2/LPP to the mutant HMGA1-binding site in the pbRho-130 mutHMGI reporter is impaired but not completely abolished. The transcriptional activation capacity of HMGA2/LPP on the wild-type pbRho-130 reporter is in any case not merely due to the LIM domains. This was concluded from experiments testing the activity of an expression vector harboring the two most carboxyl-terminal LIM domains of LPP (lacking the DNA-binding domain of HMGA2), which failed to activate the reporter (Fig. 3E). Furthermore, lack of activity of LPP-LIM2/3 was not due to its reduced expression (data not shown). Therefore, the presence of the DNA-binding domain of HMGA2 is essential for the transcriptional activity of the HMGA2/LPP fusion proteins.

HMGA2/LPP and Wild-Type HMGA2 Are Coexpressed in Lipomas
Previously, we found that HMGA2/LPP fusion transcripts are expressed in several primary human lipomas, corresponding lipoma cell lines, and a parosteal lipoma (15, 20). To determine whether wild-type HMGA2 transcripts are also expressed in these tumor-derived samples, we did reverse transcription–PCR (RT-PCR) analysis on RNA isolated from these tumor cells, with HMGA2-specific primers, which amplify the complete coding region of HMGA2. As a positive control, we used RNA that was isolated from the Hep3B cell line, which expresses high levels of HMGA2 transcripts (35). In this way, we tested eight tumor cell samples, including two lipoma cell lines (Li-14/SV40 and Li-501/SV40), five primary lipomas (2528-90, 3391-90, 444-91, 3205-91, and 568-92), and one parosteal lipoma (20). As shown in Fig. 4, three of the eight tumor cell samples that express HMGA2/LPP fusion transcripts, also express wild-type HMGA2 transcripts.



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  		FIGURE 4. HMGA2/LPP and wild-type HMGA2 are coexpressed in lipomas. RT-PCR analysis of HMGA2/LPP-positive primary human lipomas and established lipoma cell lines, using HMGA2-specific primers, shows expression of wild-type HMGA2 in three of eight samples (lanes 1, 2, and 7). Lane 9, HMGA2 expression in the Hep3B cell line, as a positive control. Lane 10, negative control reaction, to which no RNA was added.

 
The Transcriptional Activation Capacity of HMGA2/LPP Is Enhanced by Wild-Type HMGA2
Because we observed coexpression of HMGA2/LPP and wild-type HMGA2, we raised the question of whether HMGA2 might have effects on the transcriptional activity of the HMGA2/LPP fusion protein. In this study, the PRDII TKluc reporter construct was used. The results indicated that coexpression of HMGA2 significantly increased the transcriptional activity of HMGA2/LPP (Fig. 5A). Expression of only HMGA2 in the presence of the PRDII TKluc reporter construct did not increase luciferase activity (Fig. 5A). Expression of HMGA2 was verified by immunoblot analysis (Fig. 3F). We repeated the same experiments with the pbRho-130 reporter construct. Again, HMGA2 enhanced the transcriptional activity of HMGA2/LPP (Fig. 5B), whereas HMGA2 alone did not affect luciferase activity (Fig. 5B). Therefore, the transcriptional activity of the HMGA2/LPP fusion protein can be enhanced by wild-type HMGA2.



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  		FIGURE 5. The transcriptional activation capacity of HMGA2/LPP is enhanced by wild-type HMGA2. HEK 293 cells were transfected with 200 ng of PRDII TKluc reporter plasmid DNA (A) or 200 ng of pbRho-130 reporter plasmid DNA (B) together with 300 ng of pHMGA2/LPP DNA, or with 300 ng of pHMGA2/LPP DNA and 100 ng of pHMGA2 DNA (HMGA2/LPP + HMGA2), or with 100 ng of pHMGA2 DNA alone (HMGA2). The total DNA amount for transfection was kept the same in each well (600 ng) by normalizing with pcDNA3 vector DNA. Forty-eight hours after transfection, cells were harvested and assayed for luciferase activity. Activity is shown relative to basal PRDII TKluc or pbRho-130 luciferase activity (Mock). For each experiment, luciferase activity was determined in duplicate wells. Columns, means of at least three independent experiments; bars, SE.

 

   Discussion
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 Acknowledgements
 References
 
Rearrangements in the human HMGA2 gene have been found to be very common in different benign solid tumors of mesenchymal origin (13-18). Heretofore most efforts have focused on the investigation of the role of the truncated HMGA2 protein in tumorigenesis. It has been proposed that the acquisition of ectopic sequences does not increase the transforming ability of a truncated form of HMGA2, in which the acidic carboxyl-terminal tail has been separated from the AT-hooks. This would indicate that the truncation of HMGA2, rather than its fusion with functional domains of other genes, is responsible for cell transformation (36). In this study, however, we show that the in-frame fusion of LPP-derived sequences to a carboxyl-terminally truncated HMGA2 protein might play a role, perhaps in a synergistic fashion, in lipomagenesis. Indeed, we observed that the tumor-specific HMGA2/LPP fusion protein seems to be able to directly activate transcription by binding to known HMGA2 and HMGA1 DNA-binding motifs, which might result in a dramatic change in the gene expression profile of cells expressing this fusion protein. We also observed an increased activation upon coexpression of the HMGA2/LPP fusion protein with wild-type HMGA2 and showed that such coexpression actually occurs in primary human tumors.

In the literature, it has recently been shown that the acidic tail of HMGA2 is not involved in the determination of HMGA2 DNA-binding specificity, but that it can affect HMGA2/DNA complexes probably due to effects on protein-protein interaction (37). Therefore, it was suggested that HMGA2 and truncated HMGA2 (HMGA2Tr) are able to target similar genes but affect their transcription differently because of altered protein-protein interactions at promoters or enhancers (37, 38). Our results suggest that the HMGA2/LPP fusion protein is able to more directly influence gene expression profiles because of the fusion of functional domains that combines hallmark features of genuine transcription factors. This was clearly shown when only the two LIM domains of LPP were coexpressed with the reporter construct or when mutant HMGA2 binding sites were used, clearly indicating the need of also the HMGA2 DNA-binding domains to obtain transcriptional activation. Although LIM domains have a similar structural organization as zinc finger DNA binding motifs, thus far, only LIM domains of hic5 have been shown to directly interact with DNA (39). Interestingly, we observed similar effects on activation when using a reporter construct containing HMGA1-binding sites (pbRho-130).

One important aspect of the regulation of target genes that pertains to our findings are posttranslational modifications, including acetylation, methylation, and phosphorylation (40). For HMGA1, it has been suggested that removal of the acidic tail has important implications for regulatory control of its binding affinity to DNA (32). This carboxyl-terminal acidic motif contains a consensus recognition sequence for casein kinase II, which phosphorylates HMGA1 in vitro and in vivo (41). Upon casein kinase II–dependent phosphorylation of HMGA1 in vivo, its affinity for DNA decreases (42). Very recently, it was shown that HMGA2 is phosphorylated by Nek2 serine-threonine kinase in a mitogen-activated protein kinase–dependent manner, which also leads to decreased DNA affinity (43). In the latter study, the phosphorylation site(s) has not been mapped yet. However, it is clear that the carboxyl-terminal acidic motif of HMGA2 contains several serine and threonine residues that are potential phosphorylation sites (40). From these data, it might be hypothesized that a truncated HMGA2, solely consisting of the AT-hooks, will bind to certain DNA motifs with increased affinity and/or in a more unregulated fashion. Consequently, gene expression profiles in that context could be affected and this might contribute to neoplastic transformation in that way. The addition of ectopic sequences with direct transcriptional activation properties, like the LIM domains of LPP, presumably has additional consequences by affecting physiologic regulation of transcription from HMGA2 binding sites. Furthermore, binding of HMGA2/LPP to HMGA1-binding sites might further increase interference with transcription regulation, be it from HMGA1-binding sites. It has been shown that both HMGA2Tr and a chimeric HMGA2/LPP (consisting of the three AT-hooks of HMGA2 fused to the three LIM domains of LPP, i.e., an artificial fusion protein, which has never been detected in tumors, because it lacks the carboxyl-terminal part of the proline-rich region present in the long fusions) are able to transform NIH-3T3 cells without any relevant difference, with respect to transforming efficiency, growth in soft agar, tumorigenicity in nude mice, and growth rate (44). That does not exclude the possibility, however, that other cell lines require fused functional ectopic sequences to acquire a transformed phenotype and/or enhance it. Moreover, functional ectopic sequences might only exert their added effect in the complex context of an organism.

It has been suggested, based on results from studies with genetically engineered mice broadly expressing HMGA2Tr, that lipomas in such mice are a rare outcome of expression of the carboxyl-terminally truncated HMGA2 in all cells of most tissues and that, most likely, additional genetic change(s) must occur in adipose tissue before the effect of HMGA2Tr becomes evident (45). If so, it is tempting to speculate that their fusion to ectopic sequences with transcriptional activation properties, as described in this report, may account for such secondary genetic change, resulting in tumor development in vivo. Coexpression of wild-type HMGA2 could constitute an additional stimulating factor because it positively affects the transactivation capacity of the fusion protein. To address this, it would be interesting to perform comparative studies of the clinical course of primary human tumors of the same tissue type with a truncation of HMGA2 and similar tumors with a HMGA2/LPP fusion gene with or without expression of wild-type HMGA2.

The frequent fusion of truncated HMGA2 to LIM domains of LPP in primary benign human solid tumors seems to attribute a particular relevance of the acquired LPP sequences. Our results in this study also suggest that the LPP-derived sequences in HMGA2/LPP might contribute to the process of mesenchymal tumor formation by more directly affecting transcriptional regulation processes. Comparative gene expression profiling studies could be instrumental in defining the differentially expressed genes and, in this way, shed more light on the role of the HMGA2/LPP fusion protein in neoplastic cell transformation. In conclusion, our studies now reveal that the fusion of domains with transcriptional activation properties to the DNA-binding domains of HMGA2 seems to functionally transform an architectural transcription factor into a genuine transcription factor.


   Materials and Methods
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
Acknowledgements
 References
 
Plasmids
DNA manipulations were done as described by Sambrook and Russell (46). For the construction of GAL4DBD-LPP-LIM2/3 (pNW73), a DNA fragment encoding amino acids 471 to 612 of LPP was generated by PCR using oligonucleotides containing EcoRI and PstI cleavage sites (underlined; MP138: 5'-ACTGCAGAATTCAATACTCTGGAGCAGTGCAA-3'; MP4: 5'-GTCGACTGCAGCTAAAGGTCAGTGCTCGCCTTG-3') allowing cloning of this fragment into the EcoRI and PstI sites of the pM1 vector (47). As a template for the PCR, we used plasmid pMP38, which contains an LPP cDNA encompassing its complete open reading frame. For expression of native proteins in mammalian cells, sequences encoding wild-type HMGA2 or the HMGA2/LPP fusion protein were inserted into the pcDNA3 expression vector (Invitrogen, Carlsbad, CA): HMGA2 cDNA was inserted into the EcoRI site (pHMGA2) and HMGA2/LPP cDNA was inserted into the NotI site (pHMGA2/LPP). The LPP-LIM2/3 expression construct was generated by amplifying a DNA fragment encoding amino acids 471 to 612 of LPP with the use of oligonucleotides containing EcoRI and XhoI restriction sites (underlined; hLIM2F: 5'-CATGGAATTCCACCAATGGAGCAGTGCAATGTGTGTTCCAAGCC-3'; hLIM3R 5'-CATGCTCGAGCTAAAGGTCAGTGCTCGCCTTGGC-3') that allowed cloning of this fragment in the EcoRI and XhoI sites of pcDNA3.1 His A (Invitrogen).

pbRho-130 and pbRho-130 mutHMGI reporter plasmids were obtained from Prof. S.J. Ono (The Schepens Eye Research Institute, Boston, MA) and have been described before (34, 48). PRDII TKluc and mPRDII TKluc reporter plasmids were obtained from Prof. G. Manfioletti (Dipartimento di Biochimica, Biofisica e Chimica della Macromolecole, Trieste, Italy; ref. 29).

Cell Culture and Transfection
The HEK 293T and 293 cell lines (ATCC CRL-11268 and CRL-1573, respectively) were grown in DMEM/Ham's F-12 supplemented with 10% FCS. These cells were cultured at 37°C and 5% CO2.

Cells in 24-well plates were transfected using FuGENE 6 transfection reagent (Roche Applied Science, Basel, Switzerland) according to the supplier's instructions. In brief, 1.5 to 1.8 µL of FuGENE was added to 50 to 60 µL of serum-free culture medium and allowed to stand at room temperature for 5 minutes before adding it to 500 to 600 ng of DNA (experiment dependent). After 20 minutes of incubation, this transfection mixture was directly added to the cells in culture medium.

SDS-PAGE and Western Blotting
Expression of the HMGA2/LPP, HMGA2, LPP, LPP-LIM2/3, and GAL4DBD-LPP-LIM2/3 proteins was verified by transfection of corresponding expression constructs in HEK 293T cells and subsequent SDS-PAGE and Western blot analysis, as described in ref. 49. HMGA2/LPP and HMGA2 proteins were detected with the HMGA2fix antibody (38). LPP and LPP-LIM2/3 proteins were detected with the KLH-2 antibody. This antibody was prepared by immunization of New Zealand white rabbits with peptide CEDCGGLLSEGDNQG (localized in LIM3 of LPP; ref. 50). GAL4DBD-LPP-LIM2/3 protein was detected with a GAL4DBD rabbit polyclonal antibody (Tebu Bio, Le Perray en Yvelines, France).

Luciferase Reporter Assays
GAL4-Based Transactivation Experiments. Twenty-four hours upon seeding, semiconfluent HEK 293 cells on 24-well plates were cotransfected with 200 ng of a luciferase reporter construct and 150 ng DNA of a construct expressing the GAL4 DNA-binding domain (GAL4DBD) or the GAL4DBD-LPP-LIM2/3 fusion protein. The reporter construct contains the gene encoding the firefly luciferase enzyme, which is under the control of a minimal promoter containing five consecutive GAL4-binding sequences. Forty-eight hours after transfection, cells were lysed with lysis buffer [25 mmol/L glycine (pH 7.8), 15 mmol/L MgSO4, 4 mmol/L EGTA, 1 mmol/L dithiothreitol, 1% Triton X-100] and assayed for luciferase activity using the Luciferase Assay System (Promega, Madison, WI). Luciferase activity was measured in a Wallac Victor2 1420 Multilabel Counter (PerkinElmer Life Sciences, Boston, MA). For each experiment, luciferase activity was determined in duplicate wells. The results are expressed as the mean of three individual transfection experiments.

HMGA-Based Transactivation Experiments. Twenty-four hours upon seeding, semiconfluent HEK 293 cells were cotransfected with various DNA constructs, as indicated in the legends to Figs. 3 and 5. Forty-eight hours after transfection, cells were lysed and assayed for luciferase activity as described for the GAL4-based transactivation experiments.

Lipoma Cell Lines and Primary Lipomas
Cell lines used in this study included Li-14/SV40 and Li-501/SV40. The origin, chromosome aberrations, and growth conditions of these cell lines have been described before (51, 52). The primary parosteal lipoma was kindly provided by Prof. J. Bridge (University of Nebraska Medical Center, Omaha, NE). The clinical, cytogenetic and molecular characteristics of this tumor have been described before (20, 53). Primary lipomas 2528-90, 3391-90, 444-91, 3205-91, and 568-92 were kindly provided by Prof. N. Mandahl (Department of Clinical Genetics, Lund University Hospital, Lund, Sweden). Tumors were characterized by routine microscopic and histopathologic techniques and classified according to established criteria. In general, tissue specimens were quickly frozen after resection and stored at –80°C. They were all characterized by chromosome banding using standard procedures (54).

RNA Isolation and RT-PCR Analysis
Total RNA was isolated from lipoma cell lines and primary tumor samples using TRIzol Reagent (Total RNA Isolation Reagent, Invitrogen) according to the supplier's instructions. RT-PCR experiments for the detection of wild-type HMGA2 transcripts were done as follows: for first strand cDNA synthesis, 20 pmol of HMGA2-specific primer 645 (5'-TACAGCAGTTTTTCACTA-3') was mixed with 5 µg of total RNA in a volume of 12 µL. This mixture was incubated at 50°C for 5 minutes and chilled on ice. Thereafter, 4 µL of first-strand buffer [250 mmol/L Tris-HCl (pH 8.3), 375 mmol/L KCl, 15 mmol/L MgCl2], 2 µL of 0.1 mol/L dithiothreitol, 1 µL of deoxynucleotide triphosphate mix (10 mmol/L of each nucleotide), and 200 units of SuperScriptII RT (Invitrogen) were added. This mixture was incubated at 42°C for 30 minutes, whereafter 2 units of RNase H (Invitrogen) were added, and the mixture was further incubated at 55°C for 10 minutes. One microliter of this cDNA-containing mixture was subsequently used for PCR amplification. The following HMGA2-specific primers were used: TA95-3 (5'-GCGCCCCCTAGTCCTCTTCGGCAGA-3') and TA95-4 (5'-AGGCAGGATGAGCGCACGCGGTGA-3').


   Acknowledgements
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
References
 
We thank Nancy Weyns for constructing the pNW73 plasmid and Jan Brants for interesting discussions.


   Notes
Top
 Notes
Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
1 Geconcerteerde Onderzoeksacties grant 2002/10, Cancer Research Program of "Fortis Verzekeringen," Fund for Scientific Research (FWO-Vlaanderen Krediet Aan Navorsers) grant 1.5.098.03, and Belgian Federation against Cancer project A5890.

Note: M.M.R. Petit is a postdoctoral fellow of the Fund for Scientific Research, Flanders, Belgium (FWO-Vlaanderen). E.M.R. Vanoirbeek is currently in the Department of Pathophysiology, Laboratory for Skeletal Development and Joint Disorders, University of Leuven, Herestraat 49 bus 813, B-3000 Leuven, Belgium. Back

Received October 21, 2004; revised December 17, 2004; accepted January 11, 2005.


   References
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 

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